Project Summary The vestibular organs of the inner ear convey signals about head motions to the brain, resulting in motor reflexes that maintain gaze and balance as well as the perception of balance and orientation. Dysfunction of the vestibular system can therefore substantially affect the ability to lead our everyday lives. Peripheral vestibular dysfunctions, like benign paroxysmal positional vertigo (BPPV) and Meniere's disease, lead to disabling episodes of vertigo and other symptoms. To analyze the pathophysiology of such diseases, it is crucial to understand how head motion signals are processed in the vestibular peripheral organs. In the crista, the sensory organ of the semicircular canals, the sensory hair cells, respond to head rotations with a deflection of their hair bundles, activating hair cell receptor potentials. Type I hair cells are close to completely ensheathed by a postsynaptic calyx ending of the afferent vestibular nerve fiber, a unique feature of the vestibular periphery, and type II hair cells are contacted by fibers with the more conventional bouton endings. The innervation pattern of these hair cell types is quite complex, yet follows a specific morpho- physiological pattern, and results in afferent fibers with differences in their response properties, for example in their regularity of resting discharge, their response properties to external stimuli and efferent inputs. Here we investigate synaptic transmission at the highly specialized type I hair cell/calyx synapse with the aim to understand the mechanisms that underlie firing patterns of the calyx afferent fibers. We have developed a preparation of excised cristae from 2-4 week old rodents to perform electrophysiological recordings from type I hair cells and calyx afferents, for some questions simultaneously. Using confocal analysis, we also characterize the morphological features of calyx afferents and assess the localization of specific synaptic proteins using antibody labeling or live imaging with fluorescently coupled markers. In Aim 1, we characterize the relation of hair cell membrane potential and afferent firing rate. We have found that glutamate accumulation and spillover in the synaptic cleft induces slow membrane potential changes and subsequent modulation of the afferent firing rate. We investigate the contribution of release properties and glutamatergic synaptic transmission to shaping the postsynaptic response pattern. Aim 2 investigates whether a cholinergic feedback loop from the calyx to the type I hair cell exists that may modulate afferent transmission. Here we put forward a new concept, including a calyx to hair cell feedback loop that may explain some of the in vivo recorded response patterns of calyx afferent firing. In Summary, we investigate the cellular mechanisms underlying calyx afferent firing properties. These studies are designed to gain a better understanding of possible vestibular peripheral dysfunctions, a prerequisite for developing treatments for such impairments.